Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming–infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.


Introduction
In insects the degree of specificity of immune responses varies substantially, from non-specific to a high degree of specificity, even regarding closely related pathogen strains [1][2][3].There is evidence for forms of immune memory called 'immune priming', which result in enhanced survival upon infection with a pathogen, after a preliminary encounter with that pathogen or pathogen-derived cues.Immune priming could provide similar benefits for the insect host as the adaptive response does for the vertebrate host, if increased protection is specific, long-lasting, and, as mentioned in some postulations, biphasic [4][5][6][7].Cases of immune priming have been described in many insect hosts, but in most cases the exact mechanisms of these phenomena are not yet fully understood [8][9][10][11][12].
The red flour beetle Tribolium castaneum and its pathogen Bacillus thuringiensis bv.tenebrionis (Btt) provide a well-established model system to study immune priming.Feeding T. castaneum larvae with a mix of flour and filter-sterilized supernatant of sporulated Btt results in increased survival upon infection with Btt spores and toxin mix [13].This oral priming might be specific and involves the differential expression of genes targeting orally ingested pathogens [14,15].Furthermore, the gut microbiota was shown to be affected by such a treatment and essential for oral priming [16,17].
Here, we conducted mass-spectrometry-based proteomic analyses to compare the supernatant composition of Btt cultures, which induce immune priming, with those of a nonpathogenic B. thuringiensis strain, Bt407-, which do not induce immune priming.We identified the Cry3Aa toxin as a candidate elicitor and further characterized its potential involvement in oral immune priming, using priming-infection experiments.To our knowledge, this is the first study that describes proteins that may elicit oral immune priming.

(b) Preparation of priming and infection diets
We prepared spore cultures as described in [18] with some minor modifications (electronic supplementary material).For priming diets, we centrifuged grown cultures twice (2604 rcf, 15 min).We filtered the supernatants through 0.45 µm and 0.22 µm filters to remove bacterial cells and mixed the filtered supernatants (or fresh liquid medium for control diets) with 0.15 g flour ml −1 .Then we pipetted 10 µl into each well of 96-well flat bottom plates and dried them at 30°C overnight.For studying how culture age influences the ability of Btt supernatants to induce priming, we centrifuged cultures after 12 h and continuously on days 1-7, respectively, and prepared priming diets as described above.For infection diets, we centrifuged grown Btt cultures after 7 days (2604 rcf, 15 min), washed the spore pellet in 15 ml PBS, centrifuged and fetched the pellet in 5 ml PBS.We adjusted the concentration to 1 × 10 10 spores ml −1 in PBS and mixed 0.15 g flour ml −1 of the spore-PBS mix.We pipetted 10 µl into each well of 96-well flat bottom plates and dried them at 30°C overnight.

(c) Priming and infection experiments
We performed priming and infection of T. castaneum larvae as described in [18].Shortly, we individualized larvae on day 15 after oviposition onto priming diets or control diets.After 24 h we transferred the larvae onto PBS diets (0.15 g flour ml −1 PBS) and left them for 4 days at standard conditions.Finally, we transferred the larvae onto infection or PBS diets and recorded survival for 5 days.We tested all treatment levels simultaneously on each 96-well plate in an experiment and repeated the experiments in blocks (for sample size and number of blocks see electronic electronic supplementary material, file S1, supplementary method).

(d) Proteomics
To identify differentially enriched proteins in the supernatants of Btt and Bt407-cultures, we performed high resolution LC-MS/MS analysis by using an EASY-nLC 1200 (Thermo Fisher) coupled to an Exploris 480 mass spectrometer (Thermo Fisher).We separated peptides on 20 cm frit-less silica emitters (CoAnn Technologies, 0.75 µm inner diameter), packed inhouse with reversed-phase ReproSil-Pur C 18 AQ 1.9 µm resin (Dr Maisch) and kept the column constantly at 50°C.We acquired the mass spectra in data-dependent acquisition mode as outlined in Sindlinger et al. [19].We processed the raw data, by using the MaxQuant software version 2.0.3.0 [20].MS/MS spectra were assigned to a custom Btt proteome assembly (Dr Heiko Liesegang, Institute of Microbiology and Genetics, University of Göttingen, unpublished) with default settings and match between runs; LFQ and iBAQ options were enabled.For further downstream analysis we used R [21].After log 2 transformation, we imputed missing LFQ intensities based on quantile regression using impu-teLCMD.To test for differential expression, we used LIMMA [22,23].Raw data were uploaded to the JPOST repository [24].

(e) Statistical analysis
We used R [21] and RStudio [25] for statistical analyses.For survival analysis, we used Cox proportional hazards model with one random factor using the 'coxme' function from the 'coxme' package [26,27].The models therefore tested survival of the larvae according to the priming treatment as an explanatory variable, as well as either the experimental batch or the 96-well plate as a random factor [28,29].To compare the priming treatment levels, we set the control treatment (Bt medium) as the intercept, and retrieved the estimates and 95% confidence intervals (95% CI) from contrasts in the model summary.We plotted the hazard ratios (HR) of the treatment levels versus the control, with the 95% CI around the HR with 'ggplot2' [30].Final editing of the figures was done in Inkscape [31].

Results and discussion
We compared the proteomes of the supernatants of two B. thuringiensis strains, which were previously shown to differ in their ability to induce oral immune priming in T. castaneum larvae.We confirmed the described priming phenotype: exposure to supernatants of Btt led to an increased survival of T. castaneum larvae upon secondary infection with Btt spores, whereas exposure to supernatants of Bt407-did not (compared to medium control, figure 1a, electronic supplementary material, figures S3 and S4).Differential proteome profiling identified protein groups that were either uniquely expressed in one, or highly differentially expressed between Btt or Bt407-supernatants, respectively (Btt: 34 protein groups, Bt407-: 46 protein groups, figure 1b, electronic supplementary material, tables S2 and S3).
Among the most differentially expressed proteins in the Btt supernatant was the known coleopteran virulence factor Cry3Aa.Different strains of B. thuringiensis express different Cry toxins, which are specifically harmful to their respective host [32].Therefore, it might be an evolutionary advantage for the insect immune system to specifically recognize and counteract them [33].
To investigate the potential role of Cry3Aa in the process of immune priming, we used a strain that was obtained by conjugation from the naturally non-priming Bt407 strain but that carries the plasmid encoding for Cry3Aa (B.thuringiensis 407gfp-neocry+ [18] here called Bt407+), and a strain of Btt that was cured of that plasmid (Btt-).Btt supernatants induced a significant effect on survival.However, supernatants of Btt-failed to induce priming in T. castaneum larvae, whereas larvae primed with supernatants of Bt407 + showed a trend for an increased survival, always compared to fresh Bt medium (figure 2a, electronic supplementary material, figure S5 and file S2).
This indicates that Cry3Aa might be important for oral immune priming.Greenwood et al. [15] found some genes, potentially associated with the Cry toxin, upregulated in primed T. castaneum larvae, including the hexamerin gene.In other insects, this gene family helps the host resist Cry toxins either directly by interacting with the toxin or indirectly by inducing cell proliferation [34,35].Supernatant proteins that remain intact after the production of 'flour disks' for the oral priming treatment (mixing with flour, drying at 30°C for 24 h) are likely candidates.Cry toxins are known to be environmentally stable, which is also relevant for their use in commercial insecticides.Although Cry toxin crystals would not pass the filters used during preparation of the priming diet, monomers that pass the filters might be sufficient to elicit a priming response.Such monomers could accumulate early in the cultures, as the expression of Cry3Aa is sporulation-independent [36,37].Indeed, priming was evident in supernatants collected as early as 12 h after culture start compared to medium control (figure 2b, electronic supplementary material, figure S6).
It has to be noted that the Cry-carrying plasmid in the otherwise non-priming inducing bacterium (Bt407+) does not fully restore priming, suggesting that additional compounds are involved.One example in the Btt supernatant could be spore-coat-associated proteins, which may be recognized by the host as Btt-specific (figure 1b).Chitinase D, an enzyme that hydrolyses chitin in the T. castaneum peritrophic matrix, was also differentially expressed.Chitinases may contribute to B. thuringiensis toxicity [38,39], potentially synergizing with Cry3Aa [40,41].Furthermore, we cannot rule out that molecules other than proteins, like metabolites, or bacterial cell wall components such as peptidoglycans, are involved in immune priming induction.Also, proteins in the Bt407-supernatant might suppress a priming response.Proteins like immune inhibitor A and neutral protease B are more abundant in Bt407-supernatant compared to Btt supernatant (figure 1b).These proteins have been described as immunomodulatory in other organisms [42][43][44].However, we argue that a specific immune response against pathogen-associated proteins, leading to immune priming, is more likely.Our data support this as we showed that without the Cry3Aa-bearing plasmid, Btt supernatants failed to induce immune priming (figure 2a).
Our results identify the Cry toxin as a strong candidate for triggering the described oral priming response in our model system.In other organisms, sublethal doses of Cry toxins have been shown to provoke host responses like increased midgut epithelium renewal, vesicle trafficking, autophagy and apoptosis [39,[45][46][47].Such damage reactions could release 'danger' signals and thereby trigger an immune reaction [48,49].An interesting example in this context is the oral immune priming observed in mosquitoes [50], where disruption of midgut barriers by Plasmodium ookinetes enables the resident microbiota to reach the haemocoel, which in turn provokes a differentiation of haemocytes, making the host more resistant against subsequent Plasmodium infections.However, it should be noted that the specificity observed in our system may need additional explanations.Futo et al. [14] showed that two virulent strains of B. thuringiensis encoding for two different Cry toxins induced a priming response in T. castaneum in a strain-specific manner and failed to prime T. castaneum larvae against the other strain.Such specificity might be caused by differential host reactions to different Cry toxins, or via a combination of a damage response with additional specificity-conferring mechanisms.Further work is needed to test for possible connections between different Cry toxins and specific immune priming.Our study provides a basis for future work on how the insect immune system can memorize and respond specifically to certain pathogens.
Ethics.This work did not require ethical approval from a human subject or animal welfare committee.
Data accessibility.Mass spectrometry data are available via the JPOST repository: https://repository.jpostdb.org/preview/11322683365361efa5e7b7, access key 2082 [51].Additional methods, results, data and analysis code are available in the electronic supplementary material [52].
Declaration of AI use.(a) HR in T. castaneum larvae upon exposure to Btt spores after priming with filter-sterilized spore culture supernatants derived from Btt, Btt-(Btt strain cured of the Cry3Aa carrying plasmid) or Bt407+ (Bt407 strain with the Cry3Aa encoding plasmid).Btt supernatant served as the positive control for priming.There was an overall significant effect of the priming treatment on survival (χ 2 = 70.6,d.f.= 3, p ≤ 0.001) with Btt having a significant effect and Bt407 + showing a trend to induce a better survival than priming with Btt-.(b) HR caused by Btt filter-sterilized spore culture supernatant harvested at different time points during the culture of Btt versus Bt medium.Priming with Btt supernatant from each time point had a significant effect on survival to Btt (χ 2 = 75.6,d.f.= 8, p ≤ 0.001).

Figure 1 .
Figure 1.Proteomic profiles of two supernatants differing in their ability to induce immune priming.(a) Hazard ratios (HR) of T. castaneum larvae upon exposure to Btt spores after priming with Bt407-or Btt filter-sterilized spore culture supernatants (Bt407-: χ 2 = 0.6229, d.f = 1, p = 0.627; Btt: χ 2 = 41.62,d.f.= 1, p ≤ 0.001) compared to the control treatment (fresh Bt medium).Bars represent the 95% confidence intervals (95% CI).HR < 1 and no overlap of 95% CI with 1 indicate increased survival of the priming treatment.(b) Volcano plot for analysis of differential protein log 2 fold change in LFQ intensities were plotted versus Benjamini-Hochberg adjusted p-values of LIMMA.Log 2 fold change of ± 1 is indicated as vertical solid lines and an adjusted p-value of 0.05 as horizontal line.For protein groups with a log 2 fold change > 1 or < −1 and an adjusted p-value < 0.05 the product of these values is shown as an enrichment score and used to colour the respective items.hyp.: hypothetical protein.Labelled proteins: highly differentially expressed or mentioned in the text.

Figure 2 .
Figure2.Hazard ratios (HR) of T. castaneum larvae from the different levels of the priming treatment versus the control treatment (fresh Bt medium).Bars represent the 95% confidence intervals (95% CI) around the HR.HR < 1 and no overlap of 95% CI with 1 indicate an increased survival of the priming treatment.(a) HR in T. castaneum larvae upon exposure to Btt spores after priming with filter-sterilized spore culture supernatants derived from Btt, Btt-(Btt strain cured of the Cry3Aa carrying plasmid) or Bt407+ (Bt407 strain with the Cry3Aa encoding plasmid).Btt supernatant served as the positive control for priming.There was an overall significant effect of the priming treatment on survival (χ 2 = 70.6,d.f.= 3, p ≤ 0.001) with Btt having a significant effect and Bt407 + showing a trend to induce a better survival than priming with Btt-.(b) HR caused by Btt filter-sterilized spore culture supernatant harvested at different time points during the culture of Btt versus Bt medium.Priming with Btt supernatant from each time point had a significant effect on survival to Btt (χ 2 = 75.6,d.f.= 8, p ≤ 0.001).
We have not used AI-assisted technologies in creating this article.